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Lymphedema, the most common lymphatic anomaly, involves defective lymphatic valve development; yet the epigenetic modifiers underlying lymphatic valve morphogenesis remain elusive. Here, we showed that during mouse development, the histone-modifying enzyme histone deacetylase 3 (Hdac3) regulates the formation of both lymphovenous valves, which maintain the separation of the blood and lymphatic vascular systems, and the lymphatic valves. Endothelium-specific ablation of Hdac3 in mice led to blood-filled lymphatic vessels, edema, defective lymphovenous valve morphogenesis, improper lymphatic drainage, defective lymphatic valve maturation, and complete lethality. Hdac3-deficient lymphovenous valves and lymphatic vessels exhibited reduced expression of the transcription factor Gata2 and its target genes. In response to oscillatory shear stress, the transcription factors Tal1, Gata2, and Ets1/2 physically interacted with and recruited Hdac3 to the evolutionarily conserved E-box-GATA-ETS composite element of a Gata2 intragenic enhancer. In turn, Hdac3 recruited histone acetyltransferase Ep300 to form an enhanceosome complex that promoted Gata2 expression. Together, these results identify Hdac3 as a key epigenetic modifier that maintains blood-lymph separation and integrates both extrinsic forces and intrinsic cues to regulate lymphatic valve development., Introduction Thin-walled lymphatic capillaries collect interstitial fluid (lymph) and transport it via collecting lymphatic vessels to the thoracic duct, which in turn drains into the subclavian vein (1). Improper drainage [...]

Haematopoietic stem cells (HSCs) are a self-renewing population of cells that continuously replenish all blood and immune cells during the lifetime of an individual (1,2). HSCs are used clinically to treat a wide array of diseases, including acute leukaemias and congenital blood disorders, but obtaining suitable numbers of cells and finding immune-compatible donors remain serious problems. These difficulties have led to an interest in the conversion of embryonic stem cells or induced pluripotent stem cells into HSCs, which is not possible using current methodologies. To accomplish this goal, it is critical to understand the native mechanisms involved in the specification of HSCs during embryonic development. Here we demonstrate in zebrafish that Wnt16 controls a novel genetic regulatory network required for HSC specification. Non-canonical signalling by Wnt16 is required for somitic expression of the Notch ligands deltaC (dlc) and deltaD (dld), and these ligands are, in turn, required for the establishment of definitive haematopoiesis. Notch signalling downstream of Dlc and Dld is earlier than, and distinct from, known cell-autonomous requirements for Notch, strongly suggesting that novel Notch-dependent relay signal(s) induce the first HSCs in parallel to other established pathways. Our results demonstrate that somite-specific gene expression is required for the production of haemogenic endothelium., We wished to define better the role, if any, of Wnt signalling in HSC specification during embryonic development. Although Wnt signalling can exert strong effects on adult HSCs, an in [...]

The vascular endothelial growth factor (VEGF) and Notch signalling pathways play a significant role in regulating various cellular behavior and thus the role of Notch signalling in deciding the response of an endothelial cell to VGEF is described. The interaction of VEGF and Notch signalling pathways during endothelial cell differentiation and angiogenesis is discussed.

TATA-box-binding protein (TBP)-related factor 3, TRF3 (also called TBP2), is a vertebrate-specific member of the TBP family that has a conserved carboxy-terminal region and DNA-binding domain virtually identical to that [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.10.030 Byline: Van N. Pham (a), Nathan D. Lawson, Joshua W. Mugford, Louis Dye (b), Daniel Castranova (a), Brigid Lo (a), Brant M. Weinstein (a) Keywords: Zebrafish; ETS transcription factors; Intersegmental vessels; Vascular development; Angiogenesis Abstract: Members of the ETS family of transcription factors are among the first genes expressed in the developing vasculature, but loss-of-function experiments for individual ETS factors in mice have not uncovered important early functional roles for these genes. However, multiple ETS factors are expressed in spatially and temporally overlapping patterns in the developing vasculature, suggesting possible functional overlap. We have taken a comprehensive approach to exploring the function of these factors during vascular development by employing the genetic and experimental tools available in the zebrafish to analyze four ETS family members expressed together in the zebrafish vasculature; fli1, fli1b, ets1, and etsrp. We isolated and characterized an ENU-induced mutant with defects in trunk angiogenesis and positionally cloned the defective gene from this mutant, etsrp. Using the etsrp morpholinos targeting each of the four genes, we show that the four ETS factors function combinatorially during vascular and hematopoietic development. Reduction of etsrp or any of the other genes alone results in either partial or no defects in endothelial differentiation, while combined reduction in the function of all four genes causes dramatic loss of endothelial cells. Our results demonstrate that combinatorial ETS factor function is essential for early endothelial specification and differentiation. Author Affiliation: (a) Laboratory of Molecular Genetics, NICHD, NIH, Building 6B, Room 309, 6 Center Drive, Bethesda, MD 20892, USA (b) Microscopy and Imaging Core, NICHD, NIH, Bethesda, MD, USA Article History: Received 12 September 2006; Revised 17 October 2006; Accepted 20 October 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.020 Byline: Laurence Covassin (a), Julio D. Amigo (a), Kana Suzuki (b), Viktor Teplyuk (b), Juerg Straubhaar (b), Nathan D. Lawson (a) Keywords: Zebrafish; Hematopoietic; Vascular; Endothelial; Microarray Abstract: In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp).sup.y1 zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP.sup.+ versus GFP.sup.- cells isolated from Tg(fli1:egfp).sup.y1 embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP.sup.+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1.sup.+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos. Author Affiliation: (a) Program in Gene Function and Expression, University of Massachusetts Medical School, Lazare Research Building, Room 617, 364 Plantation Street, Worcester, MA 01605, USA (b) Diabetes and Endocrinology Research Center, University of Massachusetts Medical School, Biotech 2, Suite 218, 373 Plantation Street, Worcester, MA 01605, USA Article History: Received 27 April 2006; Revised 28 July 2006; Accepted 6 August 2006

Mutation of the gene encoding the Mediator component thyroid hormone receptor-associated protein (TRAP)230/MED12 affects the development of multiple systems in zebrafish embryogenesis. We isolated two ethylnitrosourea-induced alleles in the gene encoding this protein and named the locus kohtalo (kto) after the homologous locus in Drosophila. Homozygous kto mutant zebrafish embryos show defects in brain, neural crest, and kidney development and die at ~6 days postfertilization. In the affected tissues, differentiation is initiated and many cell type-specific genes are expressed, but there is a failure of morphogenesis and failure to complete differentiation. These results suggest that critical targets of TRAP230 function may include proteins important for cell mobility, cell sorting, and tissue assembly. branchial arches | Danio rerio | Mediator | morphogenesis | TRAP230

Postnatal neovascularization is essential for wound healing, cancer progression, and many other physiological functions. However, its genetic mechanism is largely unknown. In this report, we study neovascularization in regenerating adult zebrafish fins using transgenic fish that express EGFP in blood vessel endothelial cells. We first describe the morphogenesis of regenerating vessels in wild-type animals and then the phenotypic analysis of a genetic mutation that disrupts blood vessel regeneration. In wild-type zebrafish caudal fins, amputated blood vessels heal their ends by 24 h postamputation (hpa) and then reconnect arteries and veins via anastomosis, to resume blood flow at wound sites by 48 hpa. The truncated vessels regenerate by first growing excess vessels to form unstructured plexuses, resembling the primary capillary plexuses formed during embryonic vasculogenesis. Interestingly, this mode of vessel growth switches by 8 days postamputation (dpa) to growth without a plexus intermediate. During blood vessel regeneration, vessel remodeling begins during early plexus formation and continues until the original vasculature pattern is reestablished at ~35 dpa. Temperature-sensitive mutants for reg6 have profound defects in blood vessel regeneration. At the restrictive temperature, reg6 regenerating blood vessels first fail to make reconnections between severed arteries and veins, and then form enlarged vascular sinuses rather than branched vascular plexuses. Reciprocal temperature-shift experiments show that reg6 function is required throughout plexus formation, but not during later growth. Our results suggest that the reg6 mutation causes defects in branch formation and/or angiogenic sprouting. Keywords: Zebrafish; Regeneration; Angiogenesis; Plexus; Branching; Anastomosis

In this study we describe a model system that allows continuous in vivo observation of the vertebrate embryonic vasculature. We find that the zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis. We demonstrate the utility of vascular-specific transgenic zebrafish in conjunction with time-lapse multiphoton laser scanning microscopy by directly observing angiogenesis within the brain of developing embryos. Our images reveal that blood vessels undergoing active angiogenic growth display extensive filopodial activity and pathfinding behavior similar to that of neuronal growth cones. We further show, using the zebrafish mindbomb mutant as an example, that the expression of EGFP within developing blood vessels permits detailed analysis of vascular defects associated with genetic mutations. Thus, these transgenic lines allow detailed analysis of both wild type and mutant embryonic vasculature and, together with the ability to perform large scale forward-genetic screens in zebrafish, will facilitate identification of new mutants affecting vascular development. Key Words: angiogenesis; transgenics; vascular development; zebrafish.